Interpretive Summary: DNA and RNA are nucleic acids that make up the genetic blueprint of life and control chemical reactions in all cells. Monocytes are one type of white blood cell found in chickens. These monocytes can recognize bacterial DNA and viral RNA and produce chemicals that can kill bacteria and viruses. These bacteria- and virus-killing chemicals help chickens fight infections and therefore stay healthy. CpG-ODN and poly I:C are two synthetic chemicals that are similar to bacterial DNA and viral RNA, respectively. We have performed experiments to see if chicken monocytes produce bacteria- and virus-killing chemicals when they are exposed to CpG-ODN and poly I:C. We found that chicken monocytes are able to recognize CpG-ODN and poly I:C and produce bacteria- and virus-killing chemicals such as interleukins and interferons. We have also found that chicken monocytes can produce significantly more of these chemicals when they are exposed to both CpG-ODN and poly I:C at the same time. This information is important to the pharmaceutical and poultry industries in the United States because it may offer a new method of producing better vaccines for poultry and reduce the use for antibiotics.

Technical Abstract:
Toll-like receptors (TLRs) are the pattern recognition receptors of the innate immune system for various conserved pathogen-associated molecular motifs. The chicken TLR3 and TLR21 (avian equivalent to mammalian TLR9) recognize poly I:C (viral double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide), respectively. Interaction between TLR3 and TLR21 agonists poly I:C and CpG-ODN has been reported to synergize in expression of proinflammatory cytokines and chemokines and production of nitric oxide in chicken monocytes. However, the interaction between poly I:C and CpG-ODN on the expressions of interferons (IFNs) and Th1/Th2 cytokines remains unknown. The objective of the present study was to investigate the effect of the interaction between poly I:C and CpG-ODN on the mRNA expressions of IFN-a and IFN-ß, Th1 cytokines IFN-' and IL-12, Th2 cytokines IL-4, and regulatory IL-10 in chicken monocytes. When stimulated with either agonist alone, CpG-ODN was found to significantly up-regulate the expression of INF-', IL-10, and IL-12p40, but not IFN-a and IFN-ß, whereas poly I:C induced the expression of INF-', IFN-a, IFN-ß, and IL-10, but not IL-12p40. However, stimulation with a combinatory CpG-ODN and poly I:C further synergistically increased the expression of IFN-' and IL-10 mRNA. Our results provide strong evidence supporting the critical role of TLR3 and TLR21 in avian innate immunity against both viral and bacterial infections, and the synergistic interaction between the TLR3 and TLR21 pathways produces a stronger Th1-biased protective immunity in chicken monocytes. Our results also suggest a potential use of poly I:C and CpG-ODN together as more efficient adjuvant for poultry vaccine development.